Project Summary The fundamental role of the immune system is to detect self from non-self. The detection and elimination of microbial infection is critical for human survival. One challenge to the immune system is infection from an intracellular microbe because the microbe masks its presence in a host cell. One strategy of the immune system to detect microbes is the sampling of different kinds of antigens, such as peptides, lipids and glycolipids, by antigen presenting molecules. A fundamentally unique arm of the immune system is MR1, which is an antigen presenting molecule that is intracellular, ubiquitously expressed across tissues, and detects small molecules derived from microbial metabolism. These features suggest that MR1 is poised to detect intracellular microbes. MR1 presents antigens to MR1-restricted T cells. These T cells are highly prevalent in the lungs and have the ability to kill infected cells. Because MR1 presents small molecule antigens and adopts an intracellular distribution, the mechanisms governing MR1 sampling of the intracellular environment are distinct from other antigen presenting molecules. These mechanisms remain unknown. Our hypothesis is that intracellular calcium signaling is important for MR1 antigen presentation because MR1 resides in vesicles and endosomal vesicles are a source of intracellular calcium. We use Mycobacterium tuberculosis (Mtb) as a model for intracellular infection and have identified calcium-sensitive trafficking proteins and calcium channels important for MR1 antigen presentation. Aim 1 of this study will determine the relative contribution of specific calcium-sensitive Synaptotagmins to MR1 antigen presentation and we will use live-cell imaging and fluorescently tagged constructs to determine how these calcium sensitive Synaptotagmins interact with MR1 and Mtb. Aim 2 of this study will determine the relative contribution of Two-pore channels (calcium channels) to MR1 antigen presentation, interactions of these channels with MR1 and with Mtb, and we will generate an MR1 construct with a genetically encoded calcium indicator, which may allow us to determine the compartment where MR1 is loaded with antigens from Mtb.